Process for degrading mannan-containing cellulosic materials

ABSTRACT

The present invention relates to processes comprising enzymatic degradation of mannan-containing cellulosic materials for producing a hydrolyzate. The invention also relates to processes of producing a fermentation product from mannan-containing cellulosic materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/109,696filed on Jul. 5, 2016, now U.S. Pat. No. 10,287,563, which is a 35U.S.C. 371 national application of international application no. 5PCT/US2015/010423 filed Jan. 7, 2015, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application No.61/924,491 filed Jan. 7, 2014 the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processes comprising enzymaticdegradation of a mannan-containing cellulosic material for producing ahydrolyzate. The invention also relates to processes of producing afermentation product from the mannan-containing cellulosic material.

BACKGROUND OF THE INVENTION

Mannan is a non-starch polysaccharide which is a polymer of themonosaccharide mannose. Mannan is found in plant, fungal and bacterialcell walls. Mannan is present in significant amounts in certain plantresidues, such as, e.g., softwood.

Softwood is a promising feedstock for bioethanol production. Softwoodcontains up to 30% mannan in the form of galactoglucomannan.Galactoglucomannans consist of a beta-1,4 linked backbone ofbeta-D-glucopyranose and beta-D-mannopyranose units, substituted at theC-6 by alpha-D-galactopyranose units. Hemicellulose in softwood mayprevent the hydrolysis of cellulose in the absence of accessory enzymessuch as hemicellulases. For the complete hydrolysis of mannan-typehemicellulose, a wide array of enzymes is required. The main enzymeinvolved in hydrolysis of galactoglucomannan is endo-1,4-beta-mannanase(EC.3.2.1.78). Endomannanase cleaves the main chain to oligosaccharidesfacilitating the solubilisation of galactoglucomannans. The action ofendomannanases is restricted by galactose substitutions, hence theircleavage by alpha-galactosidase is needed for the complete hydrolysis ofthe polymer. Oligosaccharides from galactoglucomannan are hydrolyzed tomonomers by beta-mannosidase and beta-glucosidase.

Clarke et al. (Appl. Microbiol. Biotechnol. 53:661-667 (2000)) comparebleaching of softwood paper pulp using combinations of xylanase,mannanase, and alpha-galactosidase.

Varnai et al. (Bioresource Technology 102: 9096-9104 (2011)) disclosethat xylanase and mannanase improve the hydrolysis of softwood.

WO 2009/074685 discloses a process of hydrolyzing substrates comprisingcontacting a slurry of the mannan-containing cellulosic material with anenzyme composition comprising cellulase, mannanase, and mannosidase.

It is an object of the present invention to provide improved processesfor hydrolyzing mannan-containing cellulosic materials, e.g.,galactoglucomannan and mannan rich softwood substrates.

SUMMARY OF THE INVENTION

The present invention relates to an enzyme composition comprising one ormore endoglucanases, one or more cellobiohydrolases, one or morebeta-glucosidases, at least one beta-mannosidase, and at least onemannanase, wherein the mannanase is selected from the group consistingof Aspergillus niger mannanase, Trichoderma reesei mannanase,Corollospora maritima mannanase or Talaromyces leycettanus mannanase.

The present invention further relates to the use of such a compositionin a process for hydrolysis of a mannan-containing cellulosic materialcomprising contacting said material with said composition. In a furtherasect the present invention relates to a process for producing afermentation product, comprising:

(a) saccharifying a mannan-containing cellulosic material with an enzymecomposition of the present invention;

(b) fermenting the saccharified cellulosic material with a fermentingmicroorganism to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

In a still further aspect the present invention relates to a use of amannase and a beta-manosidase in combination with a cellulasecomposition for hydrolysing a mannan-containing cellulosic material,wherein the mannanase is selected from the group consisting ofAspergillus niger mannanase, Trichoderma reesei mannanase, Corollosporamaritima mannanase or Talaromyces leycettanus mannanase.

The present invention also relates to a process for producing afermentation product, the process comprising; a) contacting an aqueousslurry of a mannan-containing cellulosic material with an enzymecomposition of the present invention to produce a soluble hydrolyzate,and b) contacting the soluble hydrolyzate with a fermenting organism toproduce a fermentation product.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esteraseactivity can be determined using 0.5 mM p-nitrophenylacetate assubstrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esteraseis defined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-galactosidase: The term “alpha-galactosidase” means analpha-D-galactoside galactohydrolase (EC 3.2.1.22) that catalyzes thehydrolysis of terminal, non-reducing alpha-D-galactose residues inalpha-D-galactosides, including galactose oligosaccharides,galactomannans and galactolipids. Alpha-galactosidase is also known asmelibiase; alpha-D-galactosidase; alpha-galactosidase A; andalpha-galactoside galactohydrolase. Alpha-galactosidase activity can bedetermined by measuring the degradation of the colorlessp-nitrophenyl-α-D-galactopyranoside (p-NPGal) to form 4-nitrophenol,which gives a yellow color at alkaline pH that can be detected at 405nm. One alpha-galactose unit is the amount of enzyme which degrades 1mmol p-NPGal per minute under the standard conditions (37° C., pH 5.5,15 minutes).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. Alpha-glucuronidase activity can be determined according to deVries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidaseequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase.Alpha-L-arabinofuranosidase activity can be determined using 5 mg ofmedium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5in a total volume of 200 μl for 30 minutes at 40° C. followed byarabinose analysis by AMINEX® HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Auxiliary Activity 9: The term “Auxiliary Activity 9” or “AA9” means apolypeptide classified as a lytic polysaccharide monooxygenase (Quinlanet al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips etal., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20:1051-1061). AA9 polypeptides were formerly classified into the glycosidehydrolase Family 61 (GH61) according to Henrissat, 1991, Biochem. J.280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic material by anenzyme having cellulolytic activity. Cellulolytic enhancing activity canbe determined by measuring the increase in reducing sugars or theincrease of the total of cellobiose and glucose from the hydrolysis of acellulosic material by cellulolytic enzyme under the followingconditions: 1-50 mg of total protein/g of cellulose in pretreated cornstover (PCS), wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of an AA9polypeptide for 1-7 days at a suitable temperature, such as 40° C.-80°C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH,such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5,compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) andbeta-glucosidase as the source of the cellulolytic activity, wherein thebeta-glucosidase is present at a weight of at least 2-5% protein of thecellulase protein loading. In one aspect, the beta-glucosidase is anAspergillus oryzae beta-glucosidase (e.g., recombinantly produced inAspergillus oryzae according to WO 02/095014). In another aspect, thebeta-glucosidase is an Aspergillus fumigatus beta-glucosidase (e.g.,recombinantly produced in Aspergillus oryzae as described in WO02/095014).

AA9 polypeptide enhancing activity can also be determined by incubatingan AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

AA9 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic materialcatalyzed by enzyme having cellulolytic activity by reducing the amountof cellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-mannosidase: The term “beta-mannosidase” means a beta-D-mannosidemannohydrolase (EC 3.2.1.25) that catalyzes the hydrolysis of terminal,non-reducing beta-D-mannose residues in beta-D-mannosides.Beta-mannosidase is also known as beta-D-mannosidase; beta-mannosidemannohydrolase; exo-beta-D-mannanase. Mannosidase activity can bedetermined by measuring the release of p-nitrophenol (pNP) frompNP-β-mannopyranosid at 37° C. for 15 minutes. One unit of mannosidaseequals the amount of enzyme capable of releasing 1 μmole of pNP perminute from pNP-beta-mannopyranosid.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity can be determinedusing 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

Carbohydrate binding module: The term “carbohydrate binding module”means a domain within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo 1 filter paper, microcrystalline cellulose, bacterial cellulose,algal cellulose, cotton, pretreated lignocellulose, etc. The most commontotal cellulolytic activity assay is the filter paper assay usingWhatman No 1 filter paper as the substrate. The assay was established bythe International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in pretreated cornstover (PCS) (or other pretreated cellulosic material) for 3-7 days at asuitable temperature such as 40° C.-80° C., e.g., 50° C., 55° C., 60°C., 65° C., or 70° C., and a suitable pH such as 4-9, e.g., 5.0, 5.5,6.0, 6.5, or 7.0, compared to a control hydrolysis without addition ofcellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by an AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,supra). Endoglucanase activity can also be determined usingcarboxymethyl cellulose (CMC) as substrate according to the procedure ofGhose, 1987, supra, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. Feruloyl esterase activity can be determinedusing 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetatepH 5.0. One unit of feruloyl esterase equals the amount of enzymecapable of releasing 1 μmole of p-nitrophenolate anion per minute at pH5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide or domain; wherein the fragment hasbiological activity.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40-80° C., e.g., 50° C.,55° C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9, e.g.,5.0, 5.5, 6.0, 6.5, or 7.0.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Mannanase: The term “mannanase” means a mannan endo-1,4-beta-mannosidase(EC 3.2.1.78) that catalyzes the hydrolysis of beta-1,4-D-mannosidiclinkages in mannans, galactomannans and glucomannans. Mannanase is alsoknown as endo-1,4-beta-mannanase; endo-beta-1,4-mannase; beta-mannanaseB; beta-1, 4-mannan 4-mannanohydrolase; endo-beta-mannanase;beta-D-mannanase; 1,4-beta-D-mannan mannanohydrolase. Mannanase activitycan be determined by measuring the release of reducing carbohydrate fromhydrolysis of carob galactomannan. The reaction is stopped by analkaline reagent including PAHBAH amd Bi3+, which complexes withreducing sugar producing color detected at 405 nm. One unit of mannanaseequals the amount of enzyme capable of releasing 1 μmole of reducingsugar.

Mannan-containing cellulosic materials: The term “mannan-containingcellulosic material” means a cellulosic material comprising mannan. Anymannan-containing cellulosic material is contemplated according to thepresent invention. In an embodiment the mannan-containing cellulosicmaterial contains 1-25 wt. %, 2-20 wt. %, 3-15 wt. %, or 4-10 wt. %mannan. The mannan-containing cellulosic material may also compriseother constituents such as cellulosic material, including celluloseand/or hemicellulose, and may also comprise other constituents such asproteinaceous material, starch, sugars, such as fermentable sugarsand/or un-fermentable sugars.

Mannan, galacto-mannan, and galactoglucomannnan are found in plant,fungal and bacterial cell walls. Mannan-containing cellulosic materialis generally found, for example, in the stems, leaves, fruits, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. It isunderstood herein that mannan-containing cellulosic material may be inthe form of plant cell wall material containing lignin, cellulose, andhemicellulose in a mixed matrix.

The mannan-containing cellulosic material may be selected from the listconsisting of herbaceous and/or woody crops, agricultural food and feedcrops, animal feed products, tubers, roots, stems, legumes, cassavapeels, cocoa pods, rice husks and/or hulls, rice bran, cobs, straw,hulls, husks, sugar beet pulp, locust bean pulp, vegetable pomaces,agricultural crop waste, straw, stalks, leaves, corn bran, husks, cobs,rind, shells, pods, wood waste, bark, shavings, sawdust, wood pulp,pulping liquor, waste paper, cardboard, wood waste, industrial ormunicipal waste water solids, manure, by-product from brewing and/orfermentation processes, wet distillers grain, dried distillers grain,spent grain, vinasse and bagasse.

In an embodiment, the mannan-containing cellulosic material is derivedfrom softwood. Softwood is wood from gymnosperm trees such as conifers.Examples of softwood species include, but are not limited to, pines,spruces, hemlocks, firs, conifers etc., e.g., red pine (Pinus resinosa),lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Easternspruce (Picea spp.), Norway spruce (Picea abies) Douglas Fir(Pseudotsuga menziesii), Eastern Red-Cedar (Juniperos virginiana) andredwood (Sequoia sempervirens).

Softwood contains up to 30% mannans, in the form of galactoglucomannan.In softwood, the content of glucomannan increases steadily from theouter parts to the inner parts. Softwood typically contains 40-60%cellulose, 20-30% hemicellulose and 20-30% lignin. The composition afterpretreatment is very dependent on the type of pretreatment andparameters.

In an embodiment the mannan-containing cellulosic material comprisesplant material derived from an Aracaceae sp. such as Cocos mucifera,Elaeis guineensis, Elaeis malanococca, an Coffea sp., an Cyamopsis sp.such as Cyamopsis tetragonoloba (guar bean).

In one embodiment the mannan-containing cellulosic material comprisescoffee waste, guar meal, palm kernel cake, palm kernel meal and/or copracake.

In another embodiment the mannan-containing cellulosic material ismunicipal solid waste (MSW). Municipal Solid Waste (MSW) is commonlyalso known as trash, garbage, refuse or rubbish. It consists of solidwaste fractions that typically comes from municipalities and includesfor instance waste from homes, schools, offices, hospitals, institutionsetc.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. It is also known in the art thatdifferent host cells process polypeptides differently, and thus, onehost cell expressing a polynucleotide may produce a different maturepolypeptide (e.g., having a different C-terminal and/or N-terminal aminoacid) as compared to another host cell expressing the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present invention, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment.

Variant: The term “variant” means a polypeptide comprising analteration, i.e., a substitution, insertion, and/or deletion, at one ormore (e.g., several) positions. A substitution means replacement of theamino acid occupying a position with a different amino acid; a deletionmeans removal of the amino acid occupying a position; and an insertionmeans adding an amino acid adjacent to and immediately following theamino acid occupying a position.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrimann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

The present invention relates to compostions and methods/processes fordegrading mannan-containing cellulosic material using a cellulasecomposition in combination with a beta-manosidase and a mannanase. Thecellulase composition may be any suitable mixture of cellulasesnecessary to efficiently degrade cellulose. As a minimum the cellulasecomposition should contain at least three enzyme activities selectedfrom beta-glucosidase, endoglucanase, and cellobiohydrolase. Preferablythe cellobiohydrolase includes both a cellobiohydrolase I and acellobiohydrolase II. The cellulase composition may e.g. be aTrichoderma whole cellulase. A whole cellulase preparation includes allof the cellulase components naturally produced by a strain ofTrichoderma, e.g., Trichoderma reesei. The cellulase composition may inanother embodiment be a mixture of cellulases from differentmicroorganisms. For more details on these specific cellulase componentssee enzyme sections below.

Enzyme Compositions

Thus the present invention relates to a composition comprising one ormore endoglucanases, one or more cellobiohydrolases, at least onebeta-glucosidases, at least one beta-mannosidase, and at least onemannanase, wherein the mannanase is selected from the group consistingof Aspergillus niger mannanase, Trichoderma reesei mannanase, C.maritima mannanase or Talaromyces leycettanus mannanase.

AA9 Polypeptide

Any AA9 polypeptide can be used as a component of the enzymecomposition. Examples of AA9 polypeptides useful in the processes of thepresent invention include, but are not limited to, AA9 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/1449344),Myceliophthora thermophila (WO 2009/033071, WO 2009/085935, WO2009/085859, WO 2009/085864, and WO 2009/085868), Aspergillus fumigatus(WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascussp. (WO 2011/039319), Penicillium emersonii (WO 2011/041397),Thermoascus crustaceous (WO 2011/041504), Aspergillus aculeatus (WO2012/125925), Thermomyces lanuginosus (WO 2012/113340, WO 2012/129699,and WO 2012/130964), Aurantiporus alborubescens (WO 2012/122477),Trichophaea saccata (WO 2012/122477), Penicillium thomii (WO2012/122477), Talaromyces stipitatus (WO 2012/135659), Humicola insolens(WO 2012/146171), Malbranchea cinnamomea (WO 2012/101206), Talaromycesleycettanus (WO 2012/101206), Chaetomium thermophilum (WO 2012/101206),and Talaromyces emersonii (WO 2012/000892).

In one aspect, the AA9 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.,manganese or copper. In another aspect, the AA9 polypeptide is used inthe presence of a dioxy compound, a bicylic compound, a heterocycliccompound, a nitrogen-containing compound, a quinone compound, asulfur-containing compound, or a liquor obtained from a pretreatedcellulosic material such as pretreated corn stover (WO 2012/021394, WO2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO2012/021401, WO 2012/021408, and WO 2012/021410).

In an embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 1.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 2.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 3.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 4.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 5.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 6.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 7.

In another embodiment, the AA9 polypeptide has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 8.

Beta-Glucosidase

Any beta-glucosidase can be used as a component of the enzymecomposition. Examples of beta-glucosidases useful in the presentinvention include, but are not limited to, beta-glucosidases fromAspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288),Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et al.,2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO 02/095014),Penicillium brasilianum IBT 20888 (WO 2007/019442 and WO 2010/088387),Thielavia terrestris (WO 2011/035029), and Trichophaea saccata (WO2007/019442).

In an embodiment, the beta-glucosidase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 10.

In another embodiment, the beta-glucosidase has a sequence identity ofat least 70%, e.g., at least 75%, at least 80%, at least 85%, at least90%, or at least 95%, to SEQ ID NO: 11.

Beta-Mannosidase

Any beta-mannosidase can be used as a component of the enzymecomposition. Examples of beta-mannosidases useful in the presentinvention include, but are not limited to, a beta-mannosidase fromAspergillus aculeatus (SwissProt:O74168), Aspergillus niger(SwissProt:A2QWU9), Bacteroides thetaiotaomicron (SwissProt:Q8AAK6),Caenorhabditis elegans (SwissProt:Q93324), Cellulomonas fimi(SwissProt:Q9XCV4), Streptomyces sp. S27 (SwissProt:D2DFB5), Thermotogamaritima MSB8 (SwissProt:Q9X1V9), and Thermotoga neapolitana(SwissProt:Q93M25).

In an embodiment, the beta-mannosidase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 12.

Beta-Xylosidase

Any beta-xylosidase can be used as a component of the enzymecomposition. Examples of beta-xylosidases useful in the processes of thepresent invention include, but are not limited to, beta-xylosidases fromAspergillus fumigatus (WO 2011/057140), Neurospora crassa(SwissProt:Q7SOW4), Talaromyces emersonii (SwissProt:Q8X212),Talaromyces thermophilus (GeneSeqP:BAA22816), and Trichoderma reesei(UniProtKB/TrEMBL:Q92458).

In an embodiment, the beta-xylosidase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 13.

In another embodiment, the beta-xylosidase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 14.

In another embodiment, the beta-xylosidase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 33.

Cellobiohydrolase

Any cellobiohydroase I (CBH I) and cellobiohydrolase II (CBH II) can beused as a component of the enzyme composition. Examples ofcellobiohydrolases useful in the present invention include, but are notlimited to, Aspergillus aculeatus cellobiohydrolase II (WO 2011/059740),Chaetomium thermophilum cellobiohydrolase I, Chaetomium thermophilumcellobiohydrolase II, Humicola insolens cellobiohydrolase I,Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871, US2007/0238155), Penicillium occitanis cellobiohydrolase I(GenBank:AY690482), Talaromyces emersonii cellobiohydrolase I(GenBank:AF439936), Thielavia hyrcanie cellobiohydrolase II (WO2010/141325), Thielavia terrestris cellobiohydrolase II (CEL6A, WO2006/074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reeseicellobiohydrolase II, and Trichophaea saccata cellobiohydrolase II (WO2010/057086). Aspergillus fumigatus cellobiohydrolase I (WO2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO2013/028928)

In an embodiment, the cellobiohydrolase I has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 16.

In another embodiment, the cellobiohydrolase I has a sequence identityof at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, to SEQ ID NO: 18.

In an embodiment, the cellobiohydrolase II has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 15,

In another embodiment, the cellobiohydrolase II has a sequence identityof at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, to SEQ ID NO: 17.

In another embodiment, the cellobiohydrolase II has a sequence identityof at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, to SEQ ID NO: 19.

Endoglucanase

Any endoglucanase can be used as a component of the enzyme composition.In an embodiment, the endoglucanase is an endoglucanase I, endoglucanaseII, endoglucanase III, or endoglucanase V. Examples of fungalendoglucanases that can be used in the present invention, include, butare not limited to, Trichoderma reesei endoglucanase I (Penttila et al.,1986, Gene 45: 253-263), Trichoderma reesei Cel7B endoglucanase I(GenBank:M15665), Trichoderma reesei endoglucanase II (Saloheimo et al.,1988, Gene 63:11-22), Trichoderma reesei Cel5A endoglucanase II(GenBank:M19373), Trichoderma reesei endoglucanase III (Okada et al.,1988, Appl. Environ. Microbiol. 64: 555-563, GenBank:AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GenBank:Z33381), Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884),Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439), Fusarium oxysporum endoglucanase(GenBank:L29381), Humicola grisea var. thermoidea endoglucanase(GenBank:AB003107), Melanocarpus albomyces endoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase (Gen Bank:XM_324477),Humicola insolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase (WO 2007/109441, WO 2008/008070), Thermoascus aurantiacusendoglucanase I (GenBank:AF487830) and Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank: M15665). Penicillium pinophalum (WO2012/062220)

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 20.

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 21.

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 22.

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 23.

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 24.

In another embodiment, the endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 25.

Mannanase

Any mannanase can be used as a component of the enzyme composition.Mannanases have been identified in several Bacillus organisms. Forexample, Talbot et al., 1990, Appl. Environ. Microbiol. 56(11):3505-3510 describes a beta-mannanase derived from Bacillusstearothermophilus having an optimum pH of 5.5-7.5. Mendoza et al.,1994, World Journal of Microbiology and Biotechnology 10(5): 551-555describes a beta-mannanase derived from Bacillus subtilis having anoptimum activity at pH 5.0 and 55° C. JP-03047076 discloses abeta-mannanase derived from Bacillus sp., having a pH optimum of 8-10.JP-63056289 describes the production of an alkaline, thermostablebeta-mannanase. JP-08051975 discloses alkaline beta-mannanases fromalkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillusamyloliquefaciens is disclosed in WO 97/11164. WO 94/25576 discloses anenzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanaseactivity and WO 93/24622 discloses a mannanase isolated from Trichodermareesei.

The mannanase may be derived from a strain of Bacillus, such as theamino acid sequence deposited as GeneSeqP:AAY54122.

A suitable commercial mannanase preparation is Mannaway® produced byNovozymes A/S.

Other examples of mannanases include, but are not limited, mannanasesfrom Aspergillus niger (GeneSeqP: BA K16998) and Trichoderma reesei(GeneSeqP:AXQ82767).

In an embodiment, the mannanase is an Aspergillus niger mannanase,particularly the mannanase shown in SEQ ID NO: 26 or a beta-mannosidasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, or at least 95%, or at least 98% or atleast 99% to SEQ ID NO: 26. In an embodiment, the mannanase is aTrichoderma reesei mannanase, particularly the mannanase shown in SEQ IDNO: 27 or a beta-mannosidase having a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, or at least 98% or at least 99% to SEQ ID NO: 27. In anembodiment, the mannanase is a Talaromyces leycettanus mannanase,particularly the mannanase shown in SEQ ID NO: 34 or a beta-mannosidasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, or at least 95%, or at least 98% or atleast 99% to SEQ ID NO: 34. In an embodiment, the mannanase is aCorollospora maritima mannanase, particularly the mannanase shown in SEQID NO: 35 or a beta-mannosidase having a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, or at least 98% or at least 99% to SEQ ID NO: 35.

Xylanase

Any xylanase can be used as a component of the enzyme composition.Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485),Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

In an embodiment, the xylanase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 28.

In an embodiment, the xylanase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 29.

In an embodiment, the xylanase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 30.

In an embodiment, the xylanase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 31.

In an embodiment, the xylanase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 32.

Embodiments of Enzymes Compositions

In one aspect the composition comprises one or more endoglucanases, oneor more cellobiohydrolases, one or more beta-glucosidases, at least onebeta-mannosidase, and one mannanase, wherein the mannanase is selectedfrom the group consisting of Aspergillus niger mannanase, Trichodermareesei mannanase, C. maritima mannanase or Talaromyces leycettanusmannanase.

In an embodiment the composition comprises one or more endoglucanases,one or more cellobiohydrolases, one or more beta-glucosidases, at leastone beta-mannosidase, and one mannanase, wherein the beta-mannosidase isselected from A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and wherein the mannanaseis selected from A. niger mannanase shown as SEQ ID NO: 26 or a mannasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98%, or atleast 99% to SEQ ID NO: 26.

In an embodiment the composition comprises one or more endoglucanases,one or more cellobiohydrolases, one or more beta-glucosidases, at leastone beta-mannosidase, and one mannanase, wherein the beta-mannosidase isselected from A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and wherein the mannanaseis selected from T. reesei mannanase shown as SEQ ID NO: 27 or a mannasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% to SEQ ID NO: 27.

In an embodiment the composition comprises one or more endoglucanases,one or more cellobiohydrolases, one or more beta-glucosidases, at leastone beta-mannosidase, and one mannanase, wherein the beta-mannosidase isselected from A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and wherein the mannanaseis selected from C. maritima mannanase shown as SEQ ID NO: 35 or amannase having a sequence identity of at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% to SEQ ID NO: 35.

In an embodiment the composition comprises one or more endoglucanases,one or more cellobiohydrolases, one or more beta-glucosidases, at leastone beta-mannosidase, and one mannanase, wherein the beta-mannosidase isselected from A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and wherein the mannanaseis selected from T. leycettanus mannanase shown as SEQ ID NO: 34 or amannase having a sequence identity of at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% to SEQ ID NO: 34.

In a further embodiment the composition comprises an Aspergillusfumigatus GH10 xylanase (WO 2006/078256), an Aspergillus fumigatusbeta-xylosidase (WO 2011/057140), a Trichoderma reesei cellulasepreparation containing Aspergillus fumigatus cellobiohydrolase I (WO2011/057140), Aspergillus fumigatus cellobiohydrolase II (WO2011/057140), Aspergillus fumigatus beta-glucosidase variant (WO2012/044915), and Penicillium sp. (emersonii) AA9 polypeptide (WO2011/041397), and A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and a mannanase shown asSEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 or a mannasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35.

In a further embodiment the composition comprises an Aspergillusfumigatus GH10 xylanase (WO 2006/078256), an Aspergillus fumigatusbeta-xylosidase (WO 2011/057140), a Trichoderma reesei cellulasepreparation containing Aspergillus fumigatus cellobiohydrolase I (WO2011/057140), Aspergillus fumigatus cellobiohydrolase II (WO2011/057140), Aspergillus fumigatus beta-glucosidase variant (WO2012/044915), and Penicillium sp. (emersonii) AA9 polypeptide (WO2011/041397), and A. niger beta-mannosidase shown as SEQ ID NO: 12 or abeta-mannosidase having a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% to SEQ ID NO: 12, and a mannanase shown asSEQ ID NO: 34 or a mannase having a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% to SEQ ID NO: 34.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting ofan acetylmannan esterase, an acetylxylan esterase, analpha-galactosidase, an arabinanase, an arabinofuranosidase, a celluloseinducible protein (CIP), a coumaric acid esterase, an esterase, anexpansin, a feruloyl esterase, a glucuronidase, a glucuronoyl esterase,a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and aswollenin.

In one aspect, the enzyme composition comprises an acetylmannanesterase.

In another aspect, the enzyme composition comprises an acetylxylanesterase. Examples of acetylxylan esterases include, but are not limitedto, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918),Chaetomium globosum (UniProt:Q2 GWX4), Chaetomium gracile(GeneSeqP:AAB82124), Humicola insolens DSM 1800 (WO 2009/073709),Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO2010/014880), Neurospora crassa (U ni Prot: q7s259), Phaeosphaerianodorum (UniProt:Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO2009/042846).

In another aspect, the enzyme composition comprises analpha-galactosidase. Examples of alpha-galactosidases include, but arenot limited to, an alpha-galactosidase from Aspergillus aculeatus,Aspergillus niger, Emericella nidulans, and Talaromyces emersonii. In anembodiment, the alpha-galactosidase has a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 9.

In another aspect, the enzyme composition comprises an arabinanase(e.g., alpha-L-arabinanase).

In another aspect, the enzyme composition comprises anarabinofuranosidase (e.g., alpha-L-arabinofuranosidase). Examples ofarabinofuranosidases include, but are not limited to,arabinofuranosidases from Aspergillus niger (GeneSeqP:AAR94170),Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M.giganteus (WO 2006/114094).

In another aspect, the enzyme composition comprises a celluloseinducible protein (CIP).

In another aspect, the enzyme composition comprises a coumaric acidesterase.

In another aspect, the enzyme composition comprises an esterase.

In another aspect, the enzyme composition comprises an expansin.

In another aspect, the enzyme composition comprises a feruloyl esterase.Examples of feruloyl esterases (ferulic acid esterases) include, but arenot limited to, feruloyl esterases form Humicola insolens DSM 1800 (WO2009/076122), Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

In another aspect, the enzyme composition comprises a glucuronidase(e.g., alpha-D-glucuronidase). Examples of alpha-glucuronidases include,but are not limited to, alpha-glucuronidases from Aspergillus clavatus(UniProt:alcc12), Aspergillus fumigatus (SwissProt:Q4WW45), Aspergillusniger (UniProt:Q96WX9), Aspergillus terreus (SwissProt:Q0CJP9), Humicolainsolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565),Talaromyces emersonii (UniProt:Q8X211), and Trichoderma reesei(UniProt:Q99024).

In another aspect, the enzyme composition comprises a glucuronoylesterase.

In another aspect, the enzyme composition comprises a ligninolyticenzyme. In an embodiment, the ligninolytic enzyme is a manganeseperoxidase. In another embodiment, the ligninolytic enzyme is a ligninperoxidase. In another embodiment, the ligninolytic enzyme is aH₂O₂-producing enzyme.

In another aspect, the enzyme composition comprises an oxidoreductase.In an embodiment, the oxidoreductase is a catalase. In anotherembodiment, the oxidoreductase is a laccase. In another embodiment, theoxidoreductase is a peroxidase. Examples of oxidoreductases include, butare not limited to, Aspergillus lentilus catalase, Aspergillus fumigatuscatalase, Aspergillus niger catalase, Aspergillus oryzae catalase,Humicola insolens catalase, Neurospora crassa catalase, Penicilliumemersonii catalase, Scytalidium thermophilum catalase, Talaromycesstipitatus catalase, Thermoascus aurantiacus catalase, Coprinus cinereuslaccase, Myceliophthora thermophila laccase, Polyporus pinsitus laccase,Pycnoporus cinnabarinus laccase, Rhizoctonia solani laccase,Streptomyces coelicolor laccase, Coprinus cinereus peroxidase, Soyperoxidase, Royal palm peroxidase.

In another aspect, the enzyme composition comprises a pectinase.

In another aspect, the enzyme composition comprises a protease.

In another aspect, the enzyme composition comprises a swollenin.

In another aspect, the enzyme composition comprises a secondbeta-glucosidase.

In another aspect, the enzyme composition comprises a secondbeta-xylosidase.

In another aspect, the enzyme composition comprises a secondcellobiohydrolase I.

In another aspect, the enzyme composition comprises a secondcellobiohydrolase II.

In another aspect, the enzyme composition comprises a secondendoglucanase, a third endoglucanase and/or a fourth endoglucanase, eachof which may be an endoglucanase I, an endoglucanase II, anendoglucanase III, or endoglucanase V.

In another aspect, the enzyme composition comprises a second xylanase.

In another aspect, the enzyme composition comprises a Trichoderma wholecellulase composition, e.g., a Trichoderma reesei whole cellulasecomposition. A whole cellulase preparation includes all of the cellulasecomponents naturally produced by a strain of Trichoderma, e.g.,Trichoderma reesei.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more components of the enzyme composition may be native proteins,recombinant proteins, or a combination of native proteins andrecombinant proteins. For example, one or more components may be nativeproteins of a cell, which is used as a host cell to expressrecombinantly one or more other components of the enzyme composition. Itis understood herein that the recombinant proteins may be heterologousand/or native to the host cell. One or more components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes depend on several factors including,but not limited to, the mixture of cellulolytic enzymes and/orhemicellulolytic enzymes, the mannan-containing cellulosic material, theconcentration of mannan-containing cellulosic material, thepretreatment(s) of the mannan-containing cellulosic material,temperature, time, pH, and inclusion of a fermenting organism (e.g., forSimultaneous Saccharification and Fermentation).

In one aspect, an effective amount of a cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of each polypeptide to thecellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 toabout 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about1.0 mg per g of the cellulosic material.

In another aspect, an effective amount of each polypeptide tocellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g,e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, orabout 0.05 to about 0.2 g per g of cellulolytic or hemicellulolyticenzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., AA9 polypeptides can bederived or obtained from any suitable origin, including, archaeal,bacterial, fungal, yeast, plant, or animal origin. The term “obtained”also means herein that the enzyme may have been produced recombinantlyin a host organism employing methods described herein, wherein therecombinantly produced enzyme is either native or foreign to the hostorganism or has a modified amino acid sequence, e.g., having one or more(e.g., several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained by, e.g.,site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, eachpolypeptide may be a Gram-positive bacterial polypeptide having enzymeactivity, or a Gram-negative bacterial polypeptide having enzymeactivity.

Each polypeptide may also be a fungal polypeptide, e.g., a yeastpolypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides mayalso be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST® (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

The enzymes may be produced by fermentation of the above-noted microbialstrains on a nutrient medium containing suitable carbon and nitrogensources and inorganic salts, using procedures known in the art (see,e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene Manipulations inFungi, Academic Press, C A, 1991). Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Processes for Producing a Fermentation Product

The present invention also relates to a process for producing afermentation product, comprising:

(a) saccharifying a mannan-containing cellulosic material with an enzymecomposition of the invention;

(b) fermenting the saccharified cellulosic material with a fermentingmicroorganism to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

Pretreatment

The process of the present invention may further comprise pretreatingthe mannan-containing cellulosic material prior to contacting an aqueousslurry of the mannan-containing cellulosic material with an enzymecomposition of the present invention. Any pretreatment process known inthe art can be used to disrupt plant cell wall components of themannan-containing cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technology 100: 10-18; Mosier et al., 2005, BioresourceTechnology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The mannan-containing cellulosic material can also be subjected toparticle size reduction, sieving, pre-soaking, wetting, washing, and/orconditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, biological pretreatment, and sulfite cooking. Additionalpretreatments include ammonia percolation, ultrasound, electroporation,microwave, supercritical CO₂, supercritical H₂O, ozone, ionic liquid,and gamma irradiation pretreatments.

The mannan-containing cellulosic material can be pretreated beforehydrolysis and/or fermentation. Pretreatment is preferably performedprior to the hydrolysis. Alternatively, the pretreatment can be carriedout simultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the mannan-containingcellulosic material is heated to disrupt the plant cell wall components,including lignin, hemicellulose, and cellulose to make the cellulose andother fractions, e.g., hemicellulose, accessible to enzymes. Thecellulosic material is passed to or through a reaction vessel wheresteam is injected to increase the temperature to the requiredtemperature and pressure and is retained therein for the desiredreaction time. Steam pretreatment is preferably performed at 140-250°C., e.g., 160-200° C. or 170-190° C., where the optimal temperaturerange depends on optional addition of a chemical catalyst. Residencetime for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30minutes, 1-20 minutes, 3-12 minutes, or 4-10 minutes, where the optimalresidence time depends on the temperature and optional addition of achemical catalyst. Steam pretreatment allows for relatively high solidsloadings, so that the cellulosic material is generally only moist duringthe pretreatment. The steam pretreatment is often combined with anexplosive discharge of the material after the pretreatment, which isknown as steam explosion, that is, rapid flashing to atmosphericpressure and turbulent flow of the material to increase the accessiblesurface area by fragmentation (Duff and Murray, 1996, BioresourceTechnology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol.Biotechnol. 59: 618-628; U.S. Patent Application No. 2002/0164730).During steam pretreatment, hemicellulose acetyl groups are cleaved andthe resulting acid autocatalyzes partial hydrolysis of the hemicelluloseto monosaccharides and oligosaccharides. Lignin is removed to only alimited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technology 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, supra). WO 2006/110891, WO 2006/110899, WO 2006/110900,and WO 2006/110901 disclose pretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, supra, and U.S. Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosicmaterial and held at a temperature in the range of preferably 140-200°C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the mannan-containing cellulosic material is presentduring pretreatment in amounts preferably between 10-80 wt. %, e.g.,20-70 wt. % or 30-60 wt. %, such as around 40 wt. %. The pretreatedcellulosic material can be unwashed or washed using any method known inthe art, e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Sulfite cooking: This pretreatment is described in US 2011/0250638 andinvolves pretreatment of a mannan-containing cellulosic material in asulphite cooking step.

Saccharification

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed by an enzyme composition ofthe present invention to break down cellulose and/or hemicellulose tofermentable sugars, such as glucose, cellobiose, xylose, xylulose,arabinose, mannose, galactose, and/or soluble oligosaccharides. Theenzymes can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.5 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt. %, e.g., about 10 toabout 40 wt. % or about 20 to about 30 wt. %.

Fermentation

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

In one aspect, the fermenting organism comprises polynucleotidesencoding the enzymes in the enzyme composition.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolyzate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism is applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g., pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term“ketone” encompasses a substance that contains one or more ketonemoieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Processes and Uses According to the Invention

The present invention relates to processes for degradingmannan-containing cellulosic material, comprising: treating thecellulosic material with an enzyme composition of the invention. Moreparticularly the invention relates to a process for producing afermentation product, comprising:

(a) saccharifying a mannan-containing cellulosic material with an enzymecomposition of the invention;

(b) fermenting the saccharified cellulosic material with a fermentingmicroorganism to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

In another aspect the invention relates to a process for producing afermentation product, the process comprising; a) contacting an aqueousslurry of a mannan-containing cellulosic material with an enzymecomposition of the present invention to produce a soluble hydrolyzate,and b) contacting the soluble hydrolyzate with a fermenting organism toproduce a fermentation product.

In particular the fermentation product is an alcohol, more particularlyethanol.

The mannan-containing cellulosic material is in a particular embodimentselected from plant material derived from an Aracaceae sp. such as Cocosmucifera, Elaeis guineensis, Elaeis malanococca, an Coffea sp., anCyamopsis sp. such as Cyamopsis tetragonoloba (guar bean).

In one embodiment the mannan-containing cellulosic material comprisescoffee waste, guar meal, palm kernel cake, palm kernel meal and/or copracake.

In another embodiment the mannan-containing cellulosic material issoftwood.

In another embodiment the mannan-containing cellulosic material ismunicipal solid waste (MSW).

The invention further relates to a use of a mannase and abeta-manosidase in combination with a cellulase composition forhydrolysing a mannan-containing cellulosic material, wherein themannanase is selected from the group consisting of Aspergillus nigermannanase, Trichoderma reesei mannanase, C. maritima mannanase orTalaromyces leycettanus mannanase. In a particular embodiment thebeta-mannosidase is Aspergillus niger beta-mannosidase.

In the processes and uses described herein, preferably thebeta-mannosidase is selected from the beta-mannosidase shown as SEQ IDNO: 12 or a beta-mannosidase having a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% to SEQ ID NO: 12. The mannanase ispreferably selected from T. leycettanus mannanase, or the mannanaseshown as SEQ ID NO: 34 or a mannase having a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% to SEQ ID NO: 34. In anotherpreferred embodiment the mannanase is a Corollospora maritime mannanase,particularly the mannanase shown in SEQ ID NO: 35 or a beta-mannosidasehaving a sequence identity of at least 70%, e.g., at least 75%, at least80%, at least 85%, at least 90%, or at least 95%, or at least 98% or atleast 99% to SEQ ID NO: 35.

The present invention is further described by the following numberedparagraphs:

Paragraph [1]. An enzyme composition comprising one or moreendoglucanases, one or more cellobiohydrolases, one or morebeta-glucosidases, at least one beta-mannosidase, and at least onemannanase, wherein the mannanase is selected from the group consistingof Aspergillus niger mannanase, Trichoderma reesei mannanase,Corollospora maritima mannanase or Talaromyces leycettanus mannanase.Paragraph [2]. The composition of paragraph 1, wherein thebeta-mannosidase is Aspergillus niger beta-mannosidase.Paragraph [3]. The enzyme composition of paragraph 1, further comprisingone or more of an AA9 polypeptide, a beta-xylosidase, acellobiohydrolase I and a cellobiohydrolase II, or a xylanase.Paragraph [4]. The enzyme composition of paragraph 3, which comprisesthe AA9 polypeptide in an amount of 0.05-4 mg enzyme protein/g totalsolids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzyme protein/g TS.Paragraph [5]. The enzyme composition of any of paragraphs 1-4, whichcomprises the beta-glucosidase in an amount of 0.05-4 mg enzymeprotein/g total solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzymeprotein/g TS.Paragraph [6]. The enzyme composition of any of paragraphs 1-5, whichcomprises the beta-mannosidase in an amount of 0.05-4 mg enzymeprotein/g total solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzymeprotein/g TS.Paragraph [7]. The enzyme composition of any of paragraphs 1-6, whichcomprises the beta-xylosidase in an amount of 0.05-4 mg enzyme protein/gtotal solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzyme protein/g TS.Paragraph [8]. The enzyme composition of any of paragraphs 1-7, whichcomprises the cellobiohydrolase I in an amount of 0.05-4 mg enzymeprotein/g total solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzymeprotein/g TS.Paragraph [9]. The enzyme composition of any of paragraphs 1-8, whichcomprises the cellobiohydrolase II in an amount of 0.05-4 mg enzymeprotein/g total solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzymeprotein/g TS.Paragraph [10]. The enzyme composition of any of paragraphs 1-9, whichcomprises the endoglucanase in an amount of 0.05-4 mg enzyme protein/gtotal solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzyme protein/g TS.Paragraph [11]. The enzyme composition of any of paragraphs 1-10, whichcomprises the alpha-galactosidase in an amount of 0.05-4 mg enzymeprotein/g total solids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzymeprotein/g TS.Paragraph [12]. The enzyme composition of any of paragraphs 1-11, whichcomprises the mannanase in an amount of 0.05-4 mg enzyme protein/g totalsolids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzyme protein/g TS.Paragraph [13]. The enzyme composition of any of paragraphs 1-12, whichcomprises the xylanase in an amount of 0.05-4 mg enzyme protein/g totalsolids (TS), e.g., 0.1-3, 0.2-2, and 0.3-1 mg enzyme protein/g TS.Paragraph [14]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 1.Paragraph [15]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 2.Paragraph [16]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 3.Paragraph [17]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 4.Paragraph [18]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 5.Paragraph [19]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 6.Paragraph [20]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 7.Paragraph [21]. The enzyme composition of any of paragraphs 1-13,wherein the AA9 polypeptide has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 8.Paragraph [22]. The enzyme composition of any of paragraphs 1-21,wherein the beta-glucosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 10.Paragraph [23]. The enzyme composition of any of paragraphs 1-21,wherein the beta-glucosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 11.Paragraph [24]. The enzyme composition of any of paragraphs 1-23,wherein the beta-mannosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 12.Paragraph [25]. The enzyme composition of any of paragraphs 1-24,wherein the beta-xylosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 13.Paragraph [26]. The enzyme composition of any of paragraphs 1-24,wherein the beta-xylosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 14.Paragraph [27]. The enzyme composition of any of paragraphs 1-24,wherein the beta-xylosidase has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 33.Paragraph [28]. The enzyme composition of any of paragraphs 1-27,wherein the cellobiohydrolase I has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 16.Paragraph [29]. The enzyme composition of any of paragraphs 1-27,wherein the cellobiohydrolase I has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 18.Paragraph [30]. The enzyme composition of any of paragraphs 1-29,wherein the cellobiohydrolase II has a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 15.Paragraph [31]. The enzyme composition of any of paragraphs 1-29,wherein the cellobiohydrolase II has a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 17.Paragraph [32]. The enzyme composition of any of paragraphs 1-29,wherein the cellobiohydrolase II has a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 19.Paragraph [33]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase is an endoglucanase I.Paragraph [34]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase is an endoglucanase II.Paragraph [35]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase is an endoglucanase III.Paragraph [36]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase is an endoglucanase V.Paragraph [37]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 20.Paragraph [38]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 21.Paragraph [39]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 23.Paragraph [40]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 22.Paragraph [41]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 24.Paragraph [42]. The enzyme composition of any of paragraphs 1-32,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 25.Paragraph [43]. The enzyme composition of any of paragraphs 1-42,wherein the mannanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 26.Paragraph [44]. The enzyme composition of any of paragraphs 1-42,wherein the mannanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 27.Paragraph [45]. The enzyme composition of any of paragraphs 1-42,wherein the mannanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 34.Paragraph [46]. The enzyme composition of any of paragraphs 1-42,wherein the mannanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 35.Paragraph [47]. The enzyme composition of any of paragraphs 1-46,wherein the xylanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 28.Paragraph [48]. The enzyme composition of any of paragraphs 1-46,wherein the xylanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 29.Paragraph [49]. The enzyme composition of any of paragraphs 1-46,wherein the xylanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 30.Paragraph [50]. The enzyme composition of any of paragraphs 1-46,wherein the xylanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 31.Paragraph [51]. The enzyme composition of any of paragraphs 1-46,wherein the xylanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 32.Paragraph [52]. The enzyme composition of any of paragraphs 1-51,further comprising an acetylmannan esterase.Paragraph [53]. The enzyme composition of any of paragraphs 1-52,further comprising an acetylxylan esterase.Paragraph [54]. The enzyme composition of any of paragraphs 1-53,further comprising an alpha-galactosidase.Paragraph [55]. The enzyme composition of paragraph 54, wherein thealpha-galactosidase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 9.Paragraph [56]. The enzyme composition of any of paragraphs 1-55,further comprising an arabinanase (e.g., alpha-L-arabinanase).Paragraph [57]. The enzyme composition of any of paragraphs 1-56,further comprising an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase).Paragraph [58]. The enzyme composition of any of paragraphs 1-57,further comprising a cellulose inducible protein (CI P).Paragraph [59]. The enzyme composition of any of paragraphs 1-58,further comprising a coumaric acid esterase.Paragraph [60]. The enzyme composition of any of paragraphs 1-59,further comprising an esterase.Paragraph [61]. The enzyme composition of any of paragraphs 1-60,further comprising an expansin.Paragraph [62]. The enzyme composition of any of paragraphs 1-61,further comprising a feruloyl esterase.Paragraph [63]. The enzyme composition of any of paragraphs 1-62,further comprising a glucuronidase (e.g., alpha-D-glucuronidase).Paragraph [64]. The enzyme composition of any of paragraphs 1-63,further comprising a glucuronoyl esterase.Paragraph [65]. The enzyme composition of any of paragraphs 1-64,further comprising a ligninolytic enzyme.Paragraph [66]. The enzyme composition of paragraph 65, wherein theligninolytic enzyme is a manganese peroxidase.Paragraph [67]. The enzyme composition of paragraph 65, wherein theligninolytic enzyme is a lignin peroxidase.Paragraph [68]. m The enzyme composition of paragraph 65, wherein theligninolytic enzyme is a H₂O₂-producing enzyme.Paragraph [69]. The enzyme composition of any of paragraphs 1-68,further comprising an oxidoreductase.Paragraph [70]. The enzyme composition of paragraph 69, wherein theoxidoreductase is a catalase.Paragraph [71]. The enzyme composition of paragraph 69, wherein theoxidoreductase is a laccase.Paragraph [72]. The enzyme composition of paragraph 69, wherein theoxidoreductase is a peroxidase.Paragraph [73]. The enzyme composition of any of paragraphs 1-72,further comprising a pectinase.Paragraph [74]. The enzyme composition of any of paragraphs 1-73,further comprising a protease.Paragraph [75]. The enzyme composition of any of paragraphs 1-74,further comprising a swollenin.Paragraph [76]. The enzyme composition of any of paragraphs 1-75,further comprising a second beta-glucosidase (different from thebeta-glucosidase).Paragraph [77]. The enzyme composition of paragraph 76, wherein thesecond beta-glucosidase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 10.Paragraph [78]. The enzyme composition of paragraph 76, wherein thesecond beta-glucosidase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 11.Paragraph [79]. The enzyme composition of any of paragraphs 1-78,further comprising a second beta-xylosidase (different from thebeta-xylosidase).Paragraph [80]. The enzyme composition of paragraph 79, wherein thebeta-xylosidase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 13.Paragraph [81]. The enzyme composition of paragraph 79, wherein thebeta-xylosidase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 14.Paragraph [82]. The enzyme composition of paragraph 79, wherein thebeta-xylosidase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 33.Paragraph [83]. The enzyme composition of any of paragraphs 1-82,further comprising a second cellobiohydrolase I (different from thecellobiohydrolase I).Paragraph [84]. The enzyme composition of paragraph 83, wherein thesecond cellobiohydrolase I has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 16.Paragraph [85]. The enzyme composition of paragraph 84, wherein thesecond cellobiohydrolase I has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 18.Paragraph [86]. The enzyme composition of any of paragraphs 1-85,further comprising a second cellobiohydrolase II (different from thecellobiohydrolase II).Paragraph [87]. The enzyme composition of paragraph 86, wherein thesecond cellobiohydrolase II has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 15.Paragraph [88]. The enzyme composition of paragraph 86, wherein thesecond cellobiohydrolase II has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 17.Paragraph [89]. The enzyme composition of paragraph 86, wherein thesecond cellobiohydrolase II has a sequence identity of at least 70%,e.g., at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, to SEQ ID NO: 19.Paragraph [90]. The enzyme composition of any of paragraphs 1-89,further comprising a second endoglucanase, a third endoglucanase and/orfourth endoglucanase (each different from the endoglucanase and eachother).Paragraph [91]. The enzyme composition of paragraph 90, wherein thesecond endoglucanase is an endoglucanase I.Paragraph [92]. The enzyme composition of paragraph 90, wherein thesecond endoglucanase is an endoglucanase II.Paragraph [93]. The enzyme composition of paragraph 90, wherein thesecond endoglucanase is an endoglucanase III.Paragraph [94]. The enzyme composition of paragraph 90, wherein thesecond endoglucanase is an endoglucanase V.Paragraph [95]. The enzyme composition of any of paragraphs 90-94,wherein the third endoglucanase is an endoglucanase I.Paragraph [96]. The enzyme composition of any of paragraphs 90-94,wherein the third endoglucanase is an endoglucanase II.Paragraph [97]. The enzyme composition of any of paragraphs 90-94,wherein the third endoglucanase is an endoglucanase III.Paragraph [98]. The enzyme composition of any of paragraphs 90-94,wherein the third endoglucanase is an endoglucanase V.Paragraph [99]. The enzyme composition of any of paragraphs 90-98,wherein the fourth endoglucanase is an endoglucanase I.Paragraph [100]. The enzyme composition of any of paragraphs 90-98,wherein the fourth endoglucanase is an endoglucanase II.Paragraph [101]. The enzyme composition of any of paragraphs 90-98,wherein the fourth endoglucanase is an endoglucanase III.Paragraph [102]. The enzyme composition of any of paragraphs 90-98,wherein the fourth endoglucanase is an endoglucanase V.Paragraph [103]. The enzyme composition of any of paragraphs 90-102,wherein the endoglucanase has a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,to SEQ ID NO: 20, the second endoglucanase has a sequence identity of atleast 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%,or at least 95%, to SEQ ID NO: 21, the third endoglucanase has asequence identity of at least 70%, e.g., at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95%, to SEQ ID NO: 23, and thefourth endoglucanase has a sequence identity of at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%, toSEQ ID NO: 22.Paragraph [104]. The enzyme composition of any of paragraphs 1-103,further comprising a second xylanase (different from the xylanase).Paragraph [105]. The enzyme composition of paragraph 104, wherein thesecond xylanase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 28.Paragraph [106]. The enzyme composition of paragraph 104, wherein thesecond xylanase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 29.Paragraph [107]. The enzyme composition of paragraph 104, wherein thesecond xylanase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 30.Paragraph [108]. The enzyme composition of paragraph 104, wherein thesecond xylanase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 31.Paragraph [109]. The enzyme composition of paragraph 104, wherein thesecond xylanase has a sequence identity of at least 70%, e.g., at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, to SEQID NO: 32.Paragraph [110]. The enzyme composition of any of paragraphs 1-109,further comprising a Trichoderma whole cellulase composition.Paragraph [111]. The enzyme composition of paragraph 110, wherein theTrichoderma whole cellulase composition is a Trichoderma reesei wholecellulase composition.Paragraph [112]. A process for degrading a mannan-containing cellulosicmaterial, comprising: treating the cellulosic material with an enzymecomposition of any of paragraphs 1-111.Paragraph [113]. The process of paragraph 112, further comprisingpretreating the mannan-containing cellulosic material prior to treatmentof the mannan-containing cellulosic material.Paragraph [114]. The process of paragraph 113, wherein the pretreatmentis acid pretreatment carried out using an organic acid, preferablysulphuric acid, acetic acid, citric acid, tartaric acid, succinic acid,and/or mixtures thereof.Paragraph [115]. The process of any of paragraphs 112-114, furthercomprising recovering the degraded cellulosic material.Paragraph [116]. The process of paragraph 115, wherein the degradedcellulosic material is a sugar.Paragraph [117]. The process of paragraph 116, wherein the sugar isselected from the group consisting of glucose, xylose, mannose,galactose, and arabinose.Paragraph [118]. A process for producing a fermentation product,comprising:

(a) saccharifying a mannan-containing cellulosic material with an enzymecomposition of any of paragraphs 1-111;

(b) fermenting the saccharified cellulosic material with a fermentingmicroorganism to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

Paragraph [119]. The process of paragraph 118, further comprisingpretreating the mannan-containing cellulosic material prior tosaccharification.

Paragraph [120]. The process of paragraph 118 or 119, wherein thepretreatment is acid pretreatment carried out using an organic acid,preferably sulphuric acid, acetic acid, citric acid, tartaric acid,succinic acid, and/or mixtures thereof.

Paragraph [121]. The process of any of paragraphs 118-120, wherein steps(a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.

Paragraph [122]. The process of any of paragraphs 118-121, wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.

Paragraph [123]. The process of paragraph 122, wherein the fermentationproduct is ethanol.

Paragraph [124]. The process of any of paragraphs 118-123, wherein thefermenting microorganism is a yeast.

Paragraph [125]. The process of any of paragraphs 118-124, wherein thefermentation product is recovered by distillation.

Paragraph [126]. The process of any of paragraphs 112-125, wherein themannan-containing cellulosic material comprises plant material derivedfrom an Aracaceae sp. such as Cocos mucifera, Elaeis guineensis, Elaeismalanococca, an Coffea sp., an Cyamopsis sp. such as Cyamopsistetragonoloba (guar bean).Paragraph [127]. The process of any of paragraphs 112-125, wherein themannan-containing cellulosic material comprises coffee waste, guar meal,palm kernel cake, palm kernel meal and/or copra cake.Paragraph [128]. The process of any of paragraphs 112-125, wherein themannan-containing cellulosic material is softwood.Paragraph [129]. The process of any of the paragraphs 112-125, whereinthe mannan-containing cellulosic material is municipal solid waste.Paragraph [130]. A use of a mannase and a beta-manosidase in combinationwith a cellulase composition for hydrolysing a mannan-containingcellulosic material, wherein the mannanase is selected from the groupconsisting of Aspergillus niger mannanase, Trichoderma reesei mannanase,Corollospora maritima mannanase or Talaromyces leycettanus mannanase.Paragraph [131]. The use according to paragraph 130, wherein thebeta-mannosidase is Aspergillus niger beta-mannosidase, particularly thebeta-mannosidase shown in SEQ ID NO: 12 or a beta-mannosidase having asequence identity of at least 70%, e.g., at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95%, or at least 98% or at least99% to SEQ ID NO: 12.Paragraph [132]. The use according to paragraph 130 or 131, wherein themannanase is a Talaromyces leycettanus mannanase, particularly themannanase shown in SEQ ID NO: 34 or a beta-mannosidase having a sequenceidentity of at least 70%, e.g., at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, or at least 98% or at least 99% toSEQ ID NO: 34.Paragraph [133]. The use according to paragraph 130 or 131, wherein themannanase is a Corollospora maritime mannanase, particularly themannanase shown in SEQ ID NO: 35 or a beta-mannosidase having a sequenceidentity of at least 70%, e.g., at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, or at least 98% or at least 99% toSEQ ID NO: 35.Paragraph [134]. The use according to any of the paragraphs 130-133,wherein the mannan-containing cellulosic material is softwood ormunicipal solid waste.Paragraph [135]. A process for producing a fermentation product, theprocess comprising; a) contacting an aqueous slurry of amannan-containing cellulosic material with an enzyme composition of thepresent invention to produce a soluble hydrolyzate, and b) contactingthe soluble hydrolyzate with a fermenting organism to produce afermentation product.Materials & MethodsEnzymes

Cellulolytic Enzyme Composition #1: A blend of an Aspergillus fumigatusGH10 xylanase (WO 2006/078256) and Aspergillus fumigatus beta-xylosidase(WO 2011/057140) with a Trichoderma reesei cellulase preparationcontaining Aspergillus fumigatus cellobiohydrolase I (WO 2011/057140),Aspergillus fumigatus cellobiohydrolase II (WO 2011/057140), Aspergillusfumigatus beta-glucosidase variant (WO 2012/044915), and Penicillium sp.(emersonii) GH61 polypeptide (WO 2011/041397).

Cellulolytic Enzyme Composition #2: A blend of 87.67% CellulolyticEnzyme Composition #1 with 5.84% Aspergillus niger endo-mannanase (SEQID NO: 26); 3.25% Aspergillus niger beta-mannosidase (SEQ ID NO: 12);and 3.25% Aspergillus niger alpha-galactosidase (SEQ ID NO: 9).

Cellulolytic Enzyme Composition #3: A blend of 86.54% CellulolyticEnzyme Composition #1 with 8.65% Aspergillus niger endo-mannanase (SEQID NO: 26) and 4.81% Aspergillus niger beta-mannosidase (SEQ ID NO: 12).

Cellulolytic Enzyme Composition #4: A blend of 90% Cellulolytic EnzymeComposition #1 with 5% Trichoderma reesei endo-mannanase (SEQ ID NO:27); and 5% Aspergillus niger beta-mannosidase (SEQ ID NO: 12).

Cellulolytic Enzyme Composition #5: A blend of an Trichophaea saccataGH10 xylanase (WO2011/057083), Talaromyces emersonii beta-xylosidase(SwissProt:Q8X212), with a Trichoderma reesei cellulase preparationcontaining Aspergillus fumigatus cellobiohydrolase I (WO 2011/057140),Aspergillus fumigatus cellobiohydrolase II (WO 2011/057140), Aspergillusfumigatus beta-glucosidase variant (WO 2012/044915), and Penicillium sp.(emersonii) GH61 polypeptide (WO 2011/041397).

Example 1: Hydrolysis of Softwood

Wood chips from Norway spruce (Picea abies) were pretreated with steamin a two stage process as provided below. The first stage was performedat 175° C. for 30 minutes, followed by separation of the solid andliquid fraction by pressing of the wood material. The solid fractionfrom the first stage was treated in the second stage at 210° C. for 5minutes. For the hydrolysis experiments described here, only thematerial from the second stage was used. This material was mechanicallyrefined in a PFI mill according to the standard TAPPI method T248 tovarying severities of 5000 (5K) and 20000 (20K) revolutions.

Hydrolysis was performed on a 20 g scale under the following conditions:5% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hours at20 rpm in a FINEPCR Combi-D24 hybridization incubator.

Cellulolytic Enzyme Composition #1 and Cellulolytic Enzyme Composition#2 were used in the hydrolysis. The enzyme compositions were dosed at 20and 20.53 mg enzyme per g TS, respectively.

All experiments were performed in triplicate. Samples were taken after72 hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 5 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose by HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was 0.6mL/minute. Quantification was performed by integration of the glucosesignal, using a Waters 2414 Refractive index detector (50° C. in flowcell). HPLC chromatogram processing was performed using Waters Empowersoftware. HPLC data processing was performed using Microsoft Excel.Measured glucose concentrations were adjusted for appropriate dilution.

Table 1 shows the glucose concentration (g glucose/kg hydrolysis slurry)for two different substrates with Cellulolytic Enzyme Compositions #1and #2.

TABLE 1 Glucose concentration (g/kg) after 72 hours hydrolysisCellulolytic Cellulolytic Enzyme Standard Enzyme Standard SubstrateComposition #1 deviation Composition #2 deviation  5K 14.8 0.4 16.2 0.2p = 0.0095* 20K 15.0 0.5 18.1 0.3 p = 0.0014*

Cellulolytic Enzyme Composition #2 produced significantly higher glucoseconcentrations compared to Cellulolytic Enzyme Composition #1.

Example 2: Hydrolysis of Softwood

Softwood chips were pretreated in accordance with the BALI™ conceptdescribed by Borregaard in US 2011/0250638. This concept comprises asulfite cook of the softwood chips.

Hydrolysis was performed on a 20 g scale under the following conditions:10% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hoursat 20 rpm in a FINEPCR Combi-D24 hybridization incubator.

Cellulolytic Enzyme Composition #1 and Cellulolytic Enzyme Composition#2 were used in the hydrolysis. The enzyme compositions were dosed at 5,10 and 15 or 5.13, 10.27 and 15.4 mg enzyme per g TS, respectively. Allexperiments were performed in triplicate. Samples were taken after 72hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 5 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose by HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was 0.6mL/minute. Quantification was performed by integration of the glucosesignal, using a Waters 2414 Refractive index detector (50° C. in flowcell). HPLC chromatogram processing was performed using Waters Empowersoftware. HPLC data processing was performed using Microsoft Excel.Measured glucose concentrations were adjusted for appropriate dilution.

Table 2 shows the glucose concentration (g glucose/kg hydrolysis slurry)for three different enzyme doses with Cellulolytic Enzyme Compositions#1 and #2.

TABLE 2 Glucose concentration (g/kg) after 72 hours hydrolysisCellulolytic Cellulolytic Enzyme Enzyme Standard Enzyme Enzyme Standarddose Composition #1 deviation dose Composition #2 deviation 5 mg enzyme44.0 1.1 5.13 mg enzyme 52.9 1.1 p = 0.0006* per g TS per g TS 10 mgenzyme 62.4 0.9 10.27 mg enzyme 72.7 1.2 p = 0.0005* per g TS per g TS15 mg enzyme 71.2 3.9 15.4 mg enzyme 87.2 1.3 p = 0.0133* per g TS per gTS

Cellulolytic Enzyme Composition #2 produced significantly higher glucoseconcentrations compared to Cellulolytic Enzyme Composition #1.

Example 3: Hydrolysis of Softwood

Softwood chips were pretreated in accordance with the BALI™ conceptdescribed by Borregaard in US 2011/0250638.

Hydrolysis was performed on 50 g scale under the following conditions:20% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hourswith free-fall stirring in a biomass tumbler setup.

Cellulolytic Enzyme Composition #1 and Cellulolytic Enzyme Composition#2 were used in the hydrolysis experiments. The enzyme compositions weredosed at 10 and 15 or 10.27 and 15.4 mg enzyme per g TS, respectively.All experiments were performed in triplicate. Samples were taken after72 hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 5 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose by HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was 0.6mL/minute. Quantification was performed by integration of the glucosesignal, using a Waters 2414 Refractive index detector (50° C. in flowcell). HPLC chromatogram processing was performed using Waters Empowersoftware. HPLC data processing was performed using Microsoft Excel.Measured glucose concentrations were adjusted for appropriate dilution.

For mannose concentration determination, the diluted samples werecentrifuged and the supernatants were further volume/volume diluted20-fold for analysis of mannose. The samples were analyzed for glucoseand mannose with a DIONEX® BIOLC® System according to the followingmethod. Samples (10 μl) were loaded onto a DIONEX BIOLC® System equippedwith a DIONEX® CARBOPAC™ PA1 analytical column (4×250 mm) (DionexCorporation, Sunnyvale, Calif., USA) combined with a CARBOPAC™ PA1 guardcolumn (4×50 mm) (Dionex Corporation, Sunnyvale, Calif., USA). Themonosaccharides were separated isocratically with 2 mM potassiumhydroxide at a flow rate of 1 ml per minute and detected by a pulsedelectrochemical detector in the pulsed amperiometric detection mode.Mixtures of arabinose, galactose, glucose, xylose, and mannose(concentration range of each component: 0.0050-0.0750 g per liter) wereused as standards. All DIONEX chromatogram processing was performedusing Chromeleon software. All DIONEX data processing was performedusing Microsoft Excel. Measured sugar concentrations were adjusted forthe appropriate dilution factor.

Table 3 shows the glucose concentration (g glucose/kg hydrolysis slurry)for the 2 different enzyme doses with Cellulolytic Enzyme Composition #1and #2.

Table 4 shows the mannose concentration (g mannose/kg hydrolysis slurry)for the 2 different enzyme doses with Cellulolytic Enzyme Compositions#1 and #2.

TABLE 3 Glucose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 10 mgenzyme 104.9 0.7 10.27 mg enzyme 117.0 2.1 p = 0.0060* per g TS per g TS15 mg enzyme 113.2 0.6 15.4 mg enzyme 123.9 0.3 p = 0.0002* per g TS perg TS

TABLE 4 Mannose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 10 mgenzyme 4.8 0.1 10.27 mg enzyme 10.0 0.3 p = 0.0004* per g TS per g TS 15mg enzyme 6.1 0.2 15.4 mg enzyme 9.6 0.4 p = 0.0024* per g TS per g TS

Cellulolytic Enzyme Composition #2 produced significantly higher glucoseand mannose concentrations compared to Cellulolytic Enzyme Composition#1.

Example 4: Hydrolysis of Softwood

Wood chips from Norway spruce (Picea abies) were pretreated in a twostage process and then hydrolyzed as described in Example 1.

Cellulolytic Enzyme Composition #1 and Cellulolytic Enzyme Composition#2 were used in the hydrolysis experiments. The enzyme compositions weredosed at 10 or 10.27 mg enzyme per g TS, respectively. All experimentswere tested in triplicate. Samples were taken after 72 hours ofhydrolysis. The samples were deactivated at 100° C. for 10 minutes andsubsequently diluted 20-fold (weight/weight) in water. The dilutedsamples were centrifuged and the supernatants were further volume/volumediluted 20-fold and 2-fold for analysis of glucose and mannose,respectively. The samples were analyzed for glucose and mannose with aDIONEX® BIOLC® System according to the following protocol. Samples (10μl) were loaded onto a DIONEX BIOLC® System equipped with a DIONEX®CARBOPAC™ PA1 analytical column (4×250 mm) combined with a CARBOPAC™ PA1guard column (4×50 mm). The monosaccharides were separated isocraticallywith 2 mM potassium hydroxide at a flow rate of 1 ml per minute anddetected by a pulsed electrochemical detector in the pulsedamperiometric detection mode. Mixtures of arabinose, galactose, glucose,xylose and mannose (concentration range of each component: 0.0050-0.075g per liter) were used as standards. DIONEX chromatogram processing wasperformed using Chromeleon software. DIONEX data processing wasperformed using Microsoft Excel. Measured sugar concentrations wereadjusted for the appropriate dilution factor.

Table 5 shows the glucose concentration (g glucose/kg hydrolysis slurry)with Cellulolytic Enzyme Compositions #1 and #2.

Table 6 shows the mannose concentration (g mannose/kg hydrolysis slurry)with Cellulolytic Enzyme Compositions #1 and #2.

TABLE 5 Glucose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 10 mgenzyme 12.5 1.4 10.27 mg enzyme 19.0 0.3 p = 0.0103* per g TS per g TS

TABLE 6 Mannose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 10 mgenzyme 0.6 0.0 10.27 mg enzyme 2.1 0.0 p = 0.0002* per g TS per g TS

Cellulolytic Enzyme Composition #2 produced significantly higher glucoseand mannose concentrations compared to Cellulolytic Enzyme Composition#1.

Example 5: Hydrolysis of Softwood

Wood chips from Norway spruce (Picea abies) were pretreated in a twostage process as described in Example 1. The material was mechanicallyrefined in a PFI mill according to the standard TAPPI method T248 to aseverity of 20000 revolutions. For the hydrolysis experiments describedhere, the refined solid material was mixed with the liquid fraction fromthe first step of pretreatment.

Hydrolysis was performed on a 20 g scale under the following conditions:5% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hours at20 rpm in a FINEPCR Combi-D24 hybridization incubator.

Cellulolytic Enzyme Composition #1 and Cellulolytic Enzyme Composition#2 were used in the hydrolysis experiments. The enzyme compositions weredosed at 5, 10 and 20 or 5.13, 10.27 and 20.53 mg enzyme per g TS,respectively. All experiments were tested in triplicate. Samples weretaken after 72 hours of hydrolysis. The samples were deactivated at 100°C. for 10 minutes and subsequently diluted 20-fold (weight/weight) inwater. The diluted samples were centrifuged and the supernatants werefurther volume/volume diluted 20-fold and 2-fold for analysis of glucoseand mannose, respectively. The samples were analyzed for glucose, xyloseand mannose with a DIONEX® BIOLC® System according to the followingprotocol. Samples (10 μl) were loaded onto a DIONEX BIOLC® Systemequipped with a DIONEX® CARBOPAC™ PA1 analytical column (4×250 mm)combined with a CARBOPAC™ PA1 guard column (4×50 mm). Themonosaccharides were separated isocratically with 2 mM potassiumhydroxide at a flow rate of 1 ml per minute and detected by a pulsedelectrochemical detector in the pulsed amperiometric detection mode.Mixtures of arabinose, galactose, glucose, xylose and mannose(concentration range of each component: 0.0050-0.075 g per liter) wereused as standards. DIONEX chromatogram processing was performed usingChromeleon software. DIONEX data processing was performed usingMicrosoft Excel. Measured sugar concentrations were adjusted for theappropriate dilution factor.

Table 7 shows the glucose concentration (g glucose/kg hydrolysis slurry)for the three different enzyme doses with Cellulolytic EnzymeCompositions #1 and #2.

Table 8 shows the mannose concentration (g mannose/kg hydrolysis slurry)for the three different enzyme doses with Cellulolytic EnzymeCompositions #1 and #2.

TABLE 7 Glucose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 5 mgenzyme 7.2 0.1 5.13 mg enzyme 9.3 0.2 p = 0.0005* per g TS per g TS 10mg enzyme 10.2 0.7 10.27 mg enzyme 14.4 0.6 p = 0.0016* per g TS per gTS 20 mg enzyme 13.5 0.1 20.53 mg enzyme 17.4 0.1 p = 0.0001* per g TSper g TS

TABLE 8 Mannose concentration (g/kg) after 72 hours hydrolysis.Cellulolytic Cellulolytic Enzyme Enzyme Enzyme Composition StandardEnzyme Composition Standard dose #1 deviation dose #2 deviation 5 mgenzyme 1.5 0.1 5.13 mg enzyme 7.0 0.1 p = 0.0001* per g TS per g TS 10mg enzyme 2.3 0.0 10.27 mg enzyme 8.5 0.2 p = 0.0002* per g TS per g TS20 mg enzyme 4.1 0.1 20.53 mg enzyme 8.7 0.2 p = 0.0001* per g TS per gTS

Cellulolytic Enzyme Composition #2 gave significantly higher glucose andmannose concentrations compared to Cellulolytic Enzyme Composition #1.

Example 6: Hydrolysis of Softwood

Softwood chips were pretreated in accordance with the BALI™ conceptowned by Borregaard (US 2011/0250638).

Hydrolysis was performed on a 20 g scale under the following conditions:10% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hoursat 20 rpm in a FINEPCR Combi-D24 hybridization incubator.

Cellulolytic Enzyme Composition #1, Cellulolytic Enzyme Composition #3and Cellulolytic Enzyme Composition #4 were used in the hydrolysis. Theenzyme compositions were dosed at 6, 6.24 and 6 mg enzyme per g TS,respectively. All conditions were tested in triplicates. Samples weretaken after 72 hours of hydrolysis. The samples were deactivated at 100°C. for 10 min and subsequently diluted 10×(weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose on HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at column temperature of 65° C. The flow rate was 0.6mL/min. Quantification was done by integration of signals derived fromcomponents, using a Waters 2414 Refractive index detector (50° C. inflow cell). HPLC chromatogram processing was performed using WatersEmpower software. HPLC data processing was performed using MicrosoftExcel. Measured sugar concentrations were adjusted for the appropriatedilution factor.

Table 9 shows the glucose concentration (g glucose/kg hydrolysis slurry)obtained with Cellulolytic Enzyme Compositions #1 and #3.

TABLE 9 Glucose concentration (g/kg) after 72 hours hydrolysisCellulolytic Cellulolytic Enzyme Enzyme Standard Composition #3 StandardMannanase Composition #1 deviation and #4 deviation None 51.7 0.5Trichoderma 62.1 0.5 p = 0.0001* reesei (#4) Aspergillus 57.2 0.5 p =0.0002* niger (#3)

Both Cellulolytic Enzyme Composition #3 and #4 produced an increasedglucose concentration compared to Cellulolytic Enzyme Composition #1.

Example 7: Mannanases

Softwood chips were pretreated in accordance with the BALI™ conceptdescribed by Borregaard in US 2011/0250638. This concept comprises asulfite cook of the softwood chips.

Hydrolysis was performed on a 20 g scale under the following conditions:10% total solids (TS), 50 mM citrate buffer, 0.25% (w/w) of TS PEG6000,pH 5, 50° C. for 72 hours at 20 rpm in a FINEPCR Combi-D24 hybridizationincubator.

The total enzyme dose was divided between 3 components, in thepercentages shown below:

Enzyme Cellulase Condi- dose composition Beta-mannosidase Endo-mannanasetion (mg) #1 (BM) (EM) 1 6 100%  — — 2 6.12 93.1%  2.45% (A. niger 4.4%(A. niger SEQ ID NO: 12) SEQ ID NO: 26) 3 6 95% 2.5% (A. niger 2.5% (T.reesei SEQ ID NO: 12) SEQ ID NO: 27) 4 6 95% 2.5% (A. niger 2.5% (T.leycettanus SEQ ID NO: 12) SEQ ID NO: 34)

All experiments were performed in triplicate. Samples were taken after72 hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 5 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose by HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was 0.6mL/minute. Quantification was performed by integration of the glucosesignal, using a Waters 2414 Refractive index detector (50° C. in flowcell). HPLC chromatogram processing was performed using Waters Empowersoftware. HPLC data processing was performed using Microsoft Excel.Measured glucose concentrations were adjusted for appropriate dilution.

Table 10 shows the glucose concentration (g glucose/kg hydrolysisslurry) for the 4 different conditions.

TABLE 10 Glucose concentration (g/kg) after 72 hours hydrolysis Glucoseconcentration Standard Comparison with Condition (g/kg) deviationcondition 2 1 (Comp.#1) 51.7 0.5 p = 0.0002* 2 (A. niger EM) 57.2 0.5 —3 (T. reesei EM) 62.1 0.5 p = 0.0003* 4 (T. leycettanus EM) 64.1 0.2 p <0.0001*

T. leycettanus and T. reesei endo-mannanases gives significantly higherglucose concentrations compared to A. niger endo-mannanase.

Example 8: Mannanases on PKC—Thermo Stability and Conversion Efficiency

Hydrolysis of Palm Kernel Cake (PKC) was performed on a 20 g scale underthe following conditions: 25% total solids (TS), 50 mM citrate buffer,0.2% (w/w) Proxel, pH 5, 55-60-65° C. for 72 hours at 20 rpm in aFINEPCR Combi-D24 hybridization incubator.

The total enzyme dose was 0.5 mg enzyme per g TS. The total enzyme dosewas divided between 3 components, in the percentages shown below:

Cellulase Condi- Temp composition Beta-mannosidase Endo-mannanase tion °C. #1 (BM) (EM) 1 55 12% 36% (A. niger 52% (A. niger Cellulase SEQ IDNO: 12) SEQ ID NO: 26) 2 55 10% 45% (A. niger 45% (T. leycettanusCellulase SEQ ID NO: 12) SEQ ID NO: 34) 3 60 12% 36% (A. niger 52% (A.niger Cellulase SEQ ID NO: 12) SEQ ID NO: 26) 4 60 10% 45% (A. niger 45%(T. leycettanus Cellulase SEQ ID NO: 12) SEQ ID NO: 34) 5 65 12% 36% (A.niger 52% (A. niger Cellulase SEQ ID NO: 12) SEQ ID NO: 26) 6 65 10% 45%(A. niger 45% (T. leycettanus Cellulase SEQ ID NO: 12) SEQ ID NO: 34)

Prior to the trials in this example, optimal ratios between thecellulase, beta-mannosidase, A. niger/T. leycettanus endo-mannanase weredetermined.

All experiments were performed in duplicates. Samples were taken after72 hours of hydrolysis. The samples were diluted 10 times(weight/weight) in 0.005 M H₂SO₄. The diluted samples were centrifugedand HPLC samples were taken from the supernatant. The samples wereanalyzed for glucose and mannose by HPLC using a 300×7.8 mm AMINEX®HPX-87H column. Elution was isocratic using 0.005 M H₂SO₄ at a columntemperature of 65° C. The flow rate was 0.6 mL/minute. Quantificationwas performed by integration of the glucose signal, using a Waters 2414Refractive index detector (50° C. in flow cell). HPLC chromatogramprocessing was performed using Waters Empower software. HPLC dataprocessing was performed using Microsoft Excel. Measured glucose andmannose concentrations were adjusted for appropriate dilution.

Table 11 shows the glucose, mannose and average C6 sugar conversion oftheoretical possible in PKC (Conversion %) for the 6 differentconditions.

TABLE 11 Theoretical Conversion % (average) after 72 hours hydrolysis.Conversion % AVG Standard deviation Condition Temp ° C. Glucose MannoseC6 Glucose Mannose C6 1 55° C. 52.8% 70.4% 67.8% 0.55% 0.19% 0.25% 2 55°C. 59.2% 79.8% 76.8% 0.19% 0.74% 0.66% 3 60° C. 55.6% 76.2% 73.2% 2.13%0.46% 0.71% 4 60° C. 65.2% 88.4% 85.0% 1.05% 0.58% 0.34% 5 65° C. 27.7%80.3% 72.6% 2.00% 0.46% 0.68% 6 65° C. 32.3% 84.8% 77.1% 4.04% 4.02%4.02%

T. leycettanus endo-mannanase gives significantly higher averageglucose, mannose and total C6 sugar conversion compared to A. nigerendo-mannanase. Furthermore, T. leycettanus endo-mannanase showssignificantly higher thermo stability compared to A. nigerendo-mannanase.

Example 9: Mannanases on PKC—Enzyme Dosage Efficiency

Hydrolysis of Palm Kernel Cake (PKC) was performed on a 20 g scale underthe following conditions: 25% total solids (TS), 50 mM citrate buffer,0.2% (w/w) Proxel, pH 5, 60° C. for 72 hours at 20 rpm in a FINEPCRCombi-D24 hybridization incubator.

The total enzyme dose was 0.3-0.4-0.5 mg enzyme per g TS. The totalenzyme dose was divided between 3 components, in the percentages shownbelow:

Cellulase Condi- Enzyme composition Beta-mannosidase Endo-mannanase tiondosage #1 (BM) (EM) 1 0.3 12% 36% (A. niger 52% (A niger SEQ ID NO: 12)SEQ ID NO: 26) 2 0.3 10% 45% (A. niger 45% (T. leycettanus SEQ ID NO:12) SEQ ID NO: 34) 3 0.4 12% 36% (A. niger 52% (A niger SEQ ID NO: 12)SEQ ID NO: 26) 4 0.4 10% 45% (A. niger 45% (T. leycettanus SEQ ID NO:12) SEQ ID NO: 34) 5 0.5 12% 36% (A. niger 52% (A niger SEQ ID NO: 12)SEQ ID NO: 26) 6 0.5 10% 45% (A. niger 45% (T. leycettanus SEQ ID NO:12) SEQ ID NO: 34)

Prior to the trials in this example, optimal ratios between thecellulase, beta-mannosidase, A. niger/T. leycettanus endo-mannanase weredetermined.

All experiments were performed in duplicates. Samples were taken after72 hours of hydrolysis. The samples were diluted 10 times(weight/weight) in 0.005 M H₂SO₄. The diluted samples were centrifugedand HPLC samples were taken from the supernatant. The samples wereanalyzed for glucose and mannose by HPLC using a 300×7.8 mm AMINEX®HPX-87H column. Elution was isocratic using 0.005 M H₂SO₄ at a columntemperature of 65° C. The flow rate was 0.6 mL/minute. Quantificationwas performed by integration of the glucose signal, using a Waters 2414Refractive index detector (50° C. in flow cell). HPLC chromatogramprocessing was performed using Waters Empower software. HPLC dataprocessing was performed using Microsoft Excel. Measured glucose andmannose concentrations were adjusted for appropriate dilution.

Table 12 shows the glucose, mannose and average C6 sugar conversion oftheoretical possible in PKC (Conversion %) for the 6 differentconditions.

TABLE 12 Theoretical Conversion % (average) after 72 hours hydrolysis.Enzyme Conversion % AVG Standard deviation Condition dosage GlucoseMannose C6 Glucose Mannose C6 1 0.3 43.2% 68.4% 64.7% 0.22% 0.11% 0.06%2 0.3 49.1% 79.4% 75.0% 0.99% 0.74% 0.78% 3 0.4 50.6% 73.1% 69.8% 0.89%1.00% 0.99% 4 0.4 57.4% 84.0% 80.1% 0.50% 0.19% 0.23% 5 0.5 55.6% 76.2%73.2% 2.13% 0.46% 0.71% 6 0.5 65.2% 88.4% 85.0% 1.05% 0.58% 0.34%

T. leycettanus endo-mannanase gives almost same performance at 0.3 mgenzyme per g TS as A. niger endo-mannanase does at 0.5 mg enzyme per gTS. This shows that T. leycettanus endo-mannanase is significantly moreefficient than A. niger endo-mannanase at lower dosage.

Example 10: Mannanases

Softwood chips were pretreated in accordance with the BALI™ conceptdescribed by Borregaard in US 2011/0250638 A1. This concept comprises asulfite cook of the softwood chips.

Hydrolysis was performed on a 20 g scale under the following conditions:10% total solids (TS), 50 mM citrate buffer, 0.25% (w/w) of TS PEG6000,pH 5, 50° C. for 72 hours at 20 rpm in a FINEPCR Combi-D24 hybridizationincubator.

The total enzyme dose was divided between 3 components, in thepercentages shown below:

Enzyme Cellulase Condi- dose composition Beta-mannosidase Endo-mannanasetion (mg) #1 (BM) (EM) 1 6.12 93.14% 2.5% (A. niger 4.4% (A. niger SEQID NO: 12) SEQ ID NO: 26) 2 6   95% 2.5% (A. niger 2.5% (C. maritima SEQID NO: 12) SEQ ID NO: 35)

All experiments were performed in triplicate. Samples were taken after72 hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 5 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and HPLC samples were takenfrom the supernatant. The samples were analyzed for glucose by HPLCusing a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocratic using0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was 0.6mL/minute. Quantification was performed by integration of the glucosesignal, using a Waters 2414 Refractive index detector (50° C. in flowcell). HPLC chromatogram processing was performed using Waters Empowersoftware. HPLC data processing was performed using Microsoft Excel.Measured glucose concentrations were adjusted for appropriate dilution.

Table 13 shows the glucose concentration (g glucose/kg hydrolysisslurry) for the 4 different conditions.

TABLE 13 Glucose concentration (g/kg) after 72 hours hydrolysis Glucoseconcentration Standard Comparison with Condition (g/kg) deviationcondition 1 1 (A. niger EM) 52.9 1.1 — 2 (C. maritima EM) 55.7 0.6 p =0.0176*

C. maritima endo-mannanase give significantly higher glucoseconcentration compared to A. niger endo-mannanase.

Example 11: Addition of T. leycettanus Endo-Mannanase to CommercialCellulase Blend, Celluclast™, Boost Glucose and Hemicellulose Yields

Softwood chips were pretreated in accordance with the BALI™ conceptdescribed by Borregaard in US 2011/0250638. This concept comprises asulfite cook of the softwood chips. The cellulase composition used is acommercial whole cellulase blend, Celluclast™ (available from NovozymesA/S). Celluclast is a liquid cellulase preparation made by submergedfermentation of a selected strain of the fungus Trichoderma reesei.

Hydrolysis was performed on a 20 g scale under the following conditions:15% total solids (TS), 100 mM citrate buffer, 50° C., pH 5, at 20 rpm ina FINEPCR Combi-D24 hybridization incubator.

The total enzyme dose was 8.0 mg enzyme per g TS. The total enzyme dosewas divided between 3 components, in the percentages shown below:

Cellulase T. leycettanus A. niger Condi- blend endo-mannanasebeta-mannosidase tion (Celluclast ™) (EM) (BM) 1 100%  2 95% 2.5% 2.5%

All experiments were performed in quadruplicate. Samples were takenafter 72 and 96 hours of hydrolysis. The samples were deactivated at100° C. for 10 minutes and subsequently diluted 8 times (weight/weight)in HPLC eluent. The diluted samples were centrifuged and supernatantsfiltered through 0.22 μm syringe filters. The samples were analyzed forsugars by HPLC using a 300×7.8 mm AMINEX® HPX-87H column. Elution wasisocratic using 0.005 M H₂SO₄ at a column temperature of 65° C. The flowrate was 0.6 mL/minute. Quantification was performed by integration ofthe sugar signals, using a Waters 2414 Refractive index detector (50° C.in flow cell). HPLC chromatogram processing was performed using WatersEmpower software. HPLC data processing was performed using MicrosoftExcel. Measured sugar concentrations were adjusted for appropriatedilution. Hemicellulose is a measure of both mannose and xylose,quantified using xylose standards, as these two sugars co-elute on theHPX-87H column.

Table 14 shows the glucose and hemicellulose (xylose and mannose)concentration (g sugar/kg hydrolysis slurry) followed by standarddeviations in brackets for the two different conditions after differenthydrolysis times. P-values are obtained by comparison of condition 1 and2 using Student's t-test.

TABLE 14 Glucose Glucose (g/kg), (g/kg), Hemicellulose HemicelluloseCondition 72 h 96 h (g/kg), 72 h (g/kg), 96 h 1 36.35 (0.59) 41.41(0.50) 2.36 (0.03) 2.64 (0.03) 2 38.67 (0.09) 43.79 (0.38) 4.38 (0.05)4.91 (0.03) p < 0.001* p < 0.001* p < 0.0001* p < 0.0001*

Conclusion: Addition of T. leycettanus endo-mannanase and A. nigerbeta-mannosidase to Celluclast results in significantly higher glucoseand hemicellulose yields from softwood compared to Celluclast alone.

Example 12: A. niger Endo-Mannanase in Combination with CellulaseComposition #5, 20 g Scale, 5% TS at 50° C.

Wood chips from Norway spruce (Picea abies) were pretreated with steamin a two stage process. The first stage was performed at 175° C. for 30minutes, followed by separation of the solid and liquid fraction bypressing of the wood material. The solid fraction from the first stagewas treated in the second stage at 210° C. for 5 minutes. For thehydrolysis experiments described here, only the material from the secondstage was used. This material was mechanically refined in a PFI millaccording to the standard TAPPI method T248 to a severity of 20Krevolutions.

Hydrolysis was performed on a 20 g scale under the following conditions:5% total solids (TS), 50 mM citrate buffer, pH 5, 50° C. for 72 hours at20 rpm in a FINEPCR Combi-D24 hybridization incubator.

The total enzyme dose was either 10 or 20 mg Cellulase composition #5enzyme per g TS or 10.4 and 20.8 mg Cellulase composition#5/endo-mannanase/beta-mannosidase enzyme blend per g TS. The totalenzyme dose was divided between 3 components, in the percentages shownbelow:

Cellulase Beta-mannosidase Endo-mannanase Condi- composition (BM) (EM)tion #5 SEQ ID NO: 12 SEQ ID NO: 26 1  100% 0% 0% 2 86.54% 4.81% A.niger 8.65% A. niger

All experiments were performed in triplicate. Samples were taken after72 hours of hydrolysis. The samples were deactivated at 100° C. for 10minutes and subsequently diluted 10 times (weight/weight) in 0.005 MH₂SO₄. The diluted samples were centrifuged and supernatants filteredthrough 0.22 μm syringe filters. The samples were analyzed for glucoseby HPLC using a 300×7.8 mm AMINEX® HPX-87H column. Elution was isocraticusing 0.005 M H₂SO₄ at a column temperature of 65° C. The flow rate was0.6 mL/minute. Quantification was performed by integration of theglucose signal, using a Waters 2414 Refractive index detector (50° C. inflow cell). HPLC chromatogram processing was performed using WatersEmpower software. HPLC data processing was performed using MicrosoftExcel. Measured glucose concentrations were adjusted for appropriatedilution.

Table 15 shows the glucose concentration (g glucose/kg hydrolysisslurry) for the 4 different conditions.

TABLE 15 Glucose concentration (g/kg) after 72 hours hydrolysis.P-values are obtained by comparison of condition 1 and 2 using Student'st-test. Total enzyme Glucose Comparison dose concentration Standard withCondition (mg/g TS) (g/kg) deviation condition 1 1 Cellulase 10 12.170.05 — composition #5 2 Cellulase 10.4 13.17 0.09 p < 0.0001*composition #5 + EM + BM 1 Cellulase 20 16.93 0.10 — composition #5 2Cellulase 20.8 17.79 0.11 p < 0.001* composition #5 + EM + BM

Addition of A. niger endo-mannanase and beta-mannosidase to Cellulasecomposition #5 results in significantly higher glucose yields comparedto Cellulase composition #5 alone.

Example 13: Mannanases Boost Hydrolysis of MSW

Experiments were performed using different batches of Municipal SolidWaste (MSW). By supplementing cellulase composition #1 with either A.niger, T. leycettanus or T. reesei mannanase, improvements in hydrolysiswere observed.

Experiment 1:

The substrate used in this experiment was MSW substrate which was cookedand then refined in a PFI mill. Solids content was 10% and hydrolysisassays were carried out in 20 g scale at 50° C. for 72 hours. Enzymeblends and their corresponding dosage are described in table 16 as wellas the resulting glucan conversion:

TABLE 16 Mg EP/g TS Mg EP/g TS A. niger % glucan Cellulase comp. #1.mannanase conversion 3.6 0 75.6 3.42 0.32 80.1 3.24 0.65 80.2 2.88 1.3080.4Conclusion: Addition of A. niger mannanase (SEQ ID NO: 26) to Cellulasecomp. #1 boost glucan conversion of MSW.Experiment 2:

The MSW substrate used in this experiment was cooked but not refined.Solids content was 5% and hydrolysis assays were carried out in 24-wellsplates at 50° C. for 72 hours. A total protein loading of 3.6 mg enzymeprotein per gram total solids were used. Enzyme blends are described intable 17 below as well as the resulting glucan conversion:

TABLE 17 Cellulase T. leycettanus T. reesei % glucan composition # 1mannanase mannanase conversion 100%  0% 0% 31.5 95% 5% 0% 36.8 90% 0%10%  35.8Conclusion: Supplementing Cellulase composition #1 enzyme protein witheither 5% T. leycettanus mannanase (SEQ ID NO: 34) or 10% T. reeseimannanase (SEQ ID NO: 27) boost glucan conversion of MSW.

The invention claimed is:
 1. A process for degrading a mannan-containingcellulosic material, comprising: treating the mannan-containingcellulosic material with an enzyme composition comprising one or morecellulases and at least one mannanase at a temperature in the range from30° C. to 65° C., wherein the mannanase comprises SEQ ID NO: 34 or themannanase comprises an amino acid sequence having at least 90% sequenceidentity to SEQ ID NO:
 34. 2. The process of claim 1, wherein the enzymecomposition further comprises one or more of an AA9 polypeptide, abeta-glucosidase, a beta-mannosidase, a beta-xylosidase, acellobiohydrolase I, a cellobiohydrolase II, an endoglucanase, and axylanase.
 3. The process of claim 1, further comprising pretreating themannan-containing cellulosic material prior to treatment of themannan-containing cellulosic material.
 4. The process of claim 1,wherein the mannan-containing cellulosic material is municipal solidwaste.
 5. The process of claim 1, further comprising recovering thedegraded mannan-containing cellulosic material.
 6. The process of claim5, wherein the degraded mannan-containing cellulosic material is asugar.
 7. A process for producing a fermentation product, comprising:(a) saccharifying a mannan-containing cellulosic material with an enzymecomposition comprising one or more cellulases and at least one mannanaseat a temperature in the range from 30° C. to 65° C., wherein themannanase comprises SEQ ID NO: 34 or the mannanase comprises an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 34;(b) fermenting the saccharified mannan-containing cellulosic materialwith a fermenting microorganism to produce the fermentation product; and(c) recovering the fermentation product from the fermentation.
 8. Theprocess of claim 7, wherein the enzyme composition further comprises oneor more of an AA9 polypeptide, a beta-glucosidase, a beta-mannosidase, abeta-xylosidase, a cellobiohydrolase I, a cellobiohydrolase II, anendoglucanase, and a xylanase.
 9. The process of claim 7, furthercomprising pretreating the mannan-containing cellulosic material priorto saccharification.
 10. The process of claim 7, wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.
 11. A process for producing a fermentation product, theprocess comprising (a) contacting an aqueous slurry of amannan-containing cellulosic material with an enzyme composition toproduce a soluble hydrolyzate, wherein the enzyme composition comprisesone or more cellulases and at least one mannanase at a temperature inthe range from 30° C. to 65° C., wherein the mannanase comprises SEQ IDNO: 34 or the mannanase comprises an amino acid sequence having at least90% sequence identity to SEQ ID NO: 34; and (b) contacting the solublehydrolyzate with a fermenting organism to produce a fermentationproduct.
 12. The process of claim 11, wherein the enzyme compositionfurther comprises one or more of an AA9 polypeptide, a beta-glucosidase,a beta-mannosidase, a beta-xylosidase, a cellobiohydrolase I, acellobiohydrolase II, an endoglucanase, and a xylanase.